Xenobiotic-metabolizing cytochrome P450 (P450) enzymes play key roles in drug metabolism and toxin and procarcinogen activation. Since each P450 can interact with a wide range of substrates and inhibitors, understanding and predicting how foreign compounds bind and are metabolized by these enzymes is of great practical value in toxicology, chemoprevention, and polypharmacy. Building on existing knowledge in the biochemistry and structural biology of human cytochrome P450 2A and 2E1 enzymes, the objective of this proposal is to expand, test, and apply our understanding of the unique relationships between the structures of human 2A and 2E enzymes and their ligand selectivity. It is important to be able to understand the physiological and drug metabolism roles of each enzyme in order to manipulate them to positively affect human health. Relevant to this proposal, 2A13 in the respiratory track activates one of the two most prevalent and carcinogenic compounds in tobacco smoke to produce metabolites that alkylate DNA and can initiate lung cancer. Selective inhibition of 2A13 would significantly reduce this process. Specifically, we propose to 1) determine structures of human cytochrome P450 2A and 2E enzymes with physiologically relevant and marker compounds to both expand and test our understanding of ligand binding in these enzymes, 2) develop and apply a solution NMR assay of ligand binding to human membrane cytochrome P450 enzymes, and 3) identify and design potent, selective inhibitors of 2A13 for potential development as lung cancer chemopreventatives. At the completion of these studies, we expect to be able to determine if existing structures of 2A enzymes are sufficient to predict binding of new drugs and small molecules, to have proposed inhibitors to reduce tobacco- related lung cancer, and to have developed and validated solution NMR as a valuable orthogonal technique to investigate ligand binding. These results advance NIH goals indirectly by developing new methods to interrogate P450 ligand binding and directly by leading to a novel therapeutic approach for the reduction of human lung cancer. PUBLIC HEALTH RELEVANCE: This proposal investigates enzymes involved in the breakdown of drugs and the formation of carcinogens in humans. By understanding how different drugs and foreign chemicals bind and are then broken down by these enzymes, we can better predict how new drugs and known carcinogens are processed and eliminated in the human body. One specific application of this knowledge is the development of a drug that reduces the body's ability to convert nicotine into DNA-damaging molecules that can cause lung cancer in smokers.